Dynamics of endomembrane docking and fusion

内膜对接和融合的动力学

• 批准号: 7368053
• 负责人: Alexey Jarrell Merz
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7368053
• 关键词: Address; Area; Behavior; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biochemistry; Bioinformatics; Biological; Biological Assay; Biology; Cells; Chemicals; Complex; DNA Sequence Rearrangement; Dissection; Docking; Equilibrium; Event; Fluorescence Microscopy; GTP Binding; Genetic; Genomics; Goals; Green Fluorescent Proteins; Guanosine Triphosphate; Hydrolysis; In Situ; In Vitro; Individual; Intracellular Transport; Learning; Link; Lipids; Maps; Mechanics; Membrane; Membrane Biology; Methods; Microfabrication; Mitochondria; Molecular; Monitor; Nucleotides; Optics; Organelles; Pliability; Population; Probability; Proteins; Quantitative Microscopy; Reagent; Research Personnel; Resolution; Role; SNAP receptor; Saccharomyces; Saccharomycetales; Scanning; Site; System; Techniques; Testing; Touch sensation; Tube; Twin Multiple Birth; Vacuole; Work; Yeasts; analog; base; cofactor; effusion; in vitro Assay; in vivo; knowledge base; light microscopy; molecular modeling; mutant; programs; protein function; rab GTP-Binding Proteins; research study; response; sensor; size; xanthosine 5' -triphosphate

Docking and fusion events in the endomembrane system are regulated and executed by Rab GTPases, SNARE proteins, and their cofactors. A great deal has been learned about these proteins and their interactions. However, the molecular events leading to fusion are not understood in detail for any intracellular transport step. The ability to resolve individual events rather than the aggregate behavior of event populations has potentiated major advances in many areas of biology. We believe that the mechanistic dissection of membrane docking and fusion would be greatly facilitated if we could trace and perturb the subreactions of tethering, docking, and fusion at the level of individual docking and fusion events. In this proposal we combine direct attacks on the problem of monitoring single events with biochemical and genetic studies that will simultaneously characterize critical molecules and yield new reagents and probes for the single-event work. Our experimental system, the yeast vacuole, offers numerous experimental advantages for these studies including its size, which is large enough to facilitate light microscopy, the genetic and genomic toolkit of budding yeast, and a cell-free fusion system that is unsurpassed in its experimental flexibility and existing knowledge base. These experiments will build on- andsubstantially extend - approaches developed during the Pi's postdoctoral work in Dr. William Wickner's group. Microfabrication techniques and low-light, quantitative fluorescence microscopy will be; combined with the cell-free vacuole fusion system to monitor and perturb individual yeast vacuoles as they tether, dock, and fuse. We will focus our efforts on Ypt7p, the vacuole Rab, its Vps-C/HOPS effector complex, and Vam7p, a soluble SNARE. These proteins have critical functions over the entire span of the tethering-to fusion sequence, and many functional and physical interactions link them. The biological questions that we address are straightforward. Does GTP hydrolysis and exchange on the Rab Ypt7p influence the force or reversibility of tethering, or the dynamics of docking junction assembly (Aim 1)? What does the SNARE Vam7p touch as it executes its various functions in docking and fusion (Aim 2)? How is the Vps-C complex organized, and do subunits of this complex undergo structural rearrangements in response to Ypt7p-binding or other events of docking or fusion (Aim 3)? Does Ypt7p "know" when tethering, docking, or fusion have occurred (Aim 4)?

内膜系统中的对接和融合事件由 Rab GTPases 调节和执行， SNARE 蛋白及其辅助因子。人们对这些蛋白质及其作用有了很多了解 互动。然而，对于任何细胞内导致融合的分子事件尚不清楚。 运输步骤。解决单个事件而不是事件的聚合行为的能力 人口的增长促进了生物学许多领域的重大进步。我们相信机械 如果我们能够追踪和扰乱膜对接和融合的解剖将大大方便 在个体对接和融合事件水平上的系留、对接和融合的子反应。在这个 建议我们将针对单一事件监测问题的直接攻击与生化和遗传相结合 研究将同时表征关键分子并产生新的试剂和探针 单事件工作。我们的实验系统，酵母液泡，提供了许多实验优势 对于这些研究，包括它的大小，它足够大以方便光学显微镜，遗传和 芽殖酵母基因组工具包，以及实验性无与伦比的无细胞融合系统 灵活性和现有知识库。这些实验将建立在-并实质上扩展- Pi 在 William Wickner 博士小组进行博士后工作期间开发的方法。微细加工 技术和低光定量荧光显微镜；与游离液泡结合 融合系统可在单个酵母液泡束缚、对接和融合时对其进行监测和扰动。我们将重点 我们在 Ypt7p（液泡 Rab、其 Vps-C/HOPS 效应复合物）和 Vam7p（可溶性 SNARE）方面所做的努力。 这些蛋白质在束缚融合序列的整个范围内具有关键功能，并且许多 功能和物理相互作用将它们联系起来。我们要解决的生物学问题很简单。 Rab Ypt7p 上的 GTP 水解和交换是否影响束缚力或可逆性，或者 对接连接组件的动力学（目标 1）？ SNARE Vam7p 在执行其操作时会接触到什么？ 对接融合的各种功能（目标2）？ Vps-C 复合体是如何组织的，以及其子单位 该复合物响应 Ypt7p 结合或其他对接事件而发生结构重排或 融合（目标 3）？ Ypt7p 是否“知道”何时发生网络共享、对接或融合（目标 4）？